The present invention relates to a method for controllably spacing particles along an axis. More specifically, the present invention relates to a method for manipulating particles and controlling their spacing along an axis without directly contacting the particles during droplet based flow cytometry.
Piezoelectric droplet generation devices used for inkjets have been adapted for use with coulter volume and optical sensing to perform the first sorting of biological cells. The basics of this system have remained mostly unchanged since then. A hydrodynamically focused sample stream containing particles is optically interrogated (for scatter or fluorescence) either just prior or just after the focused stream is ejected into the air. The ejected stream is vibrated through the use of piezoelectric transducers, which generate waves within the sample stream leading to droplet formation. This process can lead to very regularly and predictably spaced droplets which can then be charged and deflected to be either kept for further analysis or discarded. This is one common format generally referred to as flow cytometry or often as fluorescence activated cell sorting (FACS).
However, several factors limit the speed at which this process can take place. The maximum drop formation rate requires nearly 3.45 MPa to drive 2.5×105 drops per second, though practically, mechanical limitations of the flow cytometer as well as effects on cells restrict the pressure applied to the system, limiting the rate to 1.0×105 drops per second. Additionally, because the particle arrival rate is stochastic, the maximum sort rate is additionally limited to approximately 2.5×104 particles per second. Further, the uncertain position of the particles in the flow stream leads to empty droplets, droplets with multiple particles, and particles at the break-off point between one droplet and another, all of which can lead to aborted sorting events, and all of which greatly reduce the overall sort rate.
Particle sorters of this type have problems with large particles (particles of diameter greater than 1×10−4 meters) such as multicellular organisms, multicellular spheroids, and large microspheres typically used for combinatorial library synthesis. Large particle sorters generally needed larger flow channels and exit orifices to prevent the randomly arriving particles from clogging and to prevent particle effects on droplet break-offs. However, these larger flow channels restricted the linear velocity of the flow stream due to problems caused by increased turbulence. Also, as particles took up an increasing proportion of the exit orifice, droplet formation was disturbed, effectively limiting the size of particles to about 20% the size of the exit orifice or less. Combined, these factors restricted the sorting rate of large particles to roughly 1000 per second. Some work has been done with acoustic focusing of particles, though this has not corrected the problem of random particle arrival.
Some alternative methods have been attempted, such as a high pressure air jet that deflects the flow stream unless an analysis indicates that the particles in the stream should be kept at which point the air jet shuts down allowing the particles to pass through (though this method is limited to only about 15-1000 particles per second), a fluidic switching approach which is typically mechanical in nature (limited to only about 500 particles per second), and a laser ablation approach (though this method is no faster than current techniques leaves significant cell debris and is not suitable for sorting of microspheres).
Consequently, a need has long been felt for a method of assuring predictable arrival of particles to ensure accurate high speed sorting of large particles.